1. Field of the Invention
The present invention relates to a three-dimensional position observation method and apparatus for detecting with high precision a three-dimensional position of an observation object, in particular, three-dimensional motion of a microscopic particle under a microscope.
2. Description of the Related Art
In recent years, there has been tremendous progress in optical microscopy, which now reaches a stage where a single protein molecule in an aqueous solution can be studied as an object. This progress has been achieved with the aid of new optical technologies such as total reflection illuminations, development of various types of highly sensitive cameras, improvement in properties of optical filters, and the like. A number of experimental techniques have been developed, and thus a new field called “a single molecular physiology” has emerged.
For example, in a molecular motor or a proteolytic enzyme, substrate binding involves a dynamic structural change, which is considered to be closely linked to a function.
A demanded technique is to make such a structural change occurring inside a single biomolecule visible in a molecular level in a viable condition under a microscope. Innovative techniques from a new perspective are required to advance this growing field to a next new step.
One of techniques for observing a single protein molecule is that a protein is specifically labeled with a fluorescence dye to catch a signal from a single fluorophore.
A fluorescence microscope has a structure incorporating an optical system for emitting an excitation light for brightening the fluorescence dye when receiving a light with a specific wavelength, with use of a dye emitting a light with a longer wavelength than the specific wavelength, and an optical microscope for observing thus generated fluorescence.
Where a reagent bound to a fluorescence dye is bound to a structure inside a cell as an observation object and then the fluorophore is irradiated with a light with a predetermined wavelength, the structure inside the cell as the object generates fluorescence in a black background.
Since the number of fluorophores observable with a general fluorescence microscope is several dozen or more, it is impossible to discern the single fluorophore.
This is because an optical signal strength of a noise, i.e., a background, is greater than that obtained from the single fluorophore.
In this regard, fluorescence microscopes improved for performance upgrade, with which the single fluorophore is visible, have been developed by improving a property of filters, a quality of objective lenses, and the like.
The single fluorophore is observed through the use of such a property of the fluorophore as generating fluorescence by evanescent field illumination.
Specifically, the fluorophore is made to generate fluorescence by illumination of an object sample with use of the evanescent field as a non-propagating light, which is generated around a boundary surface between an aqueous solution containing the object sample and a glass by irradiating the boundary surface with a laser beam at a total reflection angle or greater by means of total reflection from the side of the glass.
Since the evanescent field is exponentially-attenuated with respect to a direction perpendicular to the boundary surface, only a local field near the boundary surface is irradiated, thereby providing the advantage that the intensity of background light is extremely low compared with that of illumination with normal light.
Even under a condition that a number of fluorophores are present in the aqueous solution containing the object sample, there is a low probability that the fluorophore is present on the side of the aqueous solution near the boundary surface, thereby resulting in a low fluorescence from fluorophores other than the single target fluorophore secured to a top of the boundary surface. Therefore, the noise due to the fluorescence from the background light or other fluorophores is extremely low, which enables observation of the fluorescence from the single target fluorophore.
In the single-molecule observation by means of the total reflection, proteins, or biomolecule such as DNA or ATP as a substrate, which are labeled with fluorescence dyes, are bound to a glass surface to detect respective molecules as an independent bright point.
For two-dimensional imaging of a weak signal from the single molecule, a highly sensitive camera is used, such as an image intensifier or a cooled CCD camera.
The present inventors manufactured a total reflection fluorescence microscope to thereby detect a structural change of a specific part of the single biomolecule in real time by observation.
For example, “Total Reflection Fluorescence Microscope” described in Japanese Patent No. 3,577,514 relates to the basic concept and the optical system of this technique and discloses a structure of the total reflection fluorescence microscope which enables observation of a dye molecule having a vibrating surface oriented in an arbitrary direction.
“Total Reflection Type Fluorescence Microscope and Illumination Optical System” described in Japanese Patent No. 3,671,227 by the present inventors discloses a total reflection fluorescence microscope which enables observation of an target dye molecule regardless of a direction of shaking moment of a sample bound to a fluorophore.
As described above, although observation of a single biomolecule has become possible, a positional information obtainable according to the prior art is two-dimensional information. That is, the information on a vertical direction in which an objective lens moves cannot be obtained.
Observation of the three-dimensional positional information of an atomic molecule moving under a microscope achieves quantum leaps such as precise detection of displacement of the single protein molecule.
For example, the following documents also relate to the fluorescence microscope in the prior art: Japanese Patent Application Laid-Open No. 2005-37572, “Illumination Device for Fluorescence Microscope and Fluorescence Microscope”; Japanese Patent Application Laid-Open No. 2000-56233, “Device for Focusing with Adjustments Wavelength or Wavelength Region in Light Irradiation Path in Microscope”; and PCT National Publication No. 11-513145, “Confocal Microscope with Doublet System”.
There has been tremendous improvement in optical microscopy such as a bright field microscopy, a dark field microscopy, a phase difference microscopy, a differential interference microscopy, and a laser confocal microscopy. However, the positional information obtained by conventional microscopic observation is two-dimensional information in a surface (an x-y plane) parallel to a slide glass corresponding to a viewing plane, and positional information on a direction (a z-axis) perpendicular to the aforementioned surface cannot be obtained.